Antigen Transduced T Cells Used as a Delivery System for Antigens

ABSTRACT

A delivery system comprising a T cell comprising at least one antigen capable of loading antigen-presenting-cells with the antigen.

FIELD OF THE INVENTION

The present invention relates to a delivery system for an antigen in theform of a T cell, the use of such a T cell to monitor an immune responseand a method of preparing the T cell.

BACKGROUND OF THE INVENTION

Gene transfer into human cells is being investigated for the treatmentof a variety of genetic and acquired human diseases. Genetic diseases,due to a single-gene defect, were originally proposed as the primarytargets of gene therapy. However, the majority of the human gene therapytrials approved so far involve treatment of acquired diseases, suchcancer and AIDS. One approach to tumor gene therapy relies on thestimulation of the host immune system against tumor antigens byvaccination of the patient with genetically modified tumor cells, inwhich the transgene (cytokine, allo-HLA, etc.) is aimed at boostingtheir immunogenicity. An alternative approach is based on the adoptivetransfer of ex vivo generated tumor-specific effectors. The infusedeffectors are usually autologous or HLA-compatible lymphocytesstimulated in vitro to proliferate by specific as well as non-specificstimuli. Amplification of the effector function of the infused cells hasbeen achieved by their transduction with cytokine-encoding genes.

EP 0 904 786 discloses a method of tumor vaccination with at least twosubsequent administrations of a compositions consisting essentially ofautologous or HLA-related cells which are capable of presenting antigenin a patient, said cells being either tumor cells transduced with aforeign gene or antigen presenting cells transduced with a tumor antigenand a foreign antigen.

WO97/19169 discloses a tumor vaccine containing tumor cells at least aportion of which has at least one MHC-I haplotype of the patient on thecell surface, and which have been loaded with an antigen.

Thus, the prior art takes the approach of using a tumor cell as thedelivery system for an antigen, or an antigen presenting cell which isunderstood as presenting the antigen directly to a T cell.

It will be however appreciated that there is a continuing need toprovide vaccines and vaccination approaches. The present inventionprovides such an approach.

SUMMARY OF THE INVENTION

The present invention relates to manipulated T cells that whenintroduced into a patient are able to bring antigens, such as tumor,bacterial or viral antigens, into contact with professional antigenpresenting cells (APCs). More particularly, the invention provides atransduced T cell containing an exogenous (or foreign) gene encoding anantigen to stimulate a response against the antigen, and T cell loadedwith an antigenic polypeptide to stimulate a response. The T cell mayalso comprise a further antigen such as HSV-Tk which may be used tomonitor vaccination effectiveness. Conveniently the T cells are preparedin vitro and when injected, or otherwise introduced in vivo, carry thetransgene products to professional antigen presenting cells, thusstimulating the immune response against the transgene product. Theinvention also provides a methodology whereby one can monitor theimmunological status of the patient. The present invention furtherprovides a culturing method for T cells.

Thus, we have now found that T cells containing an antigen act to bringthe antigen to an antigen presenting cell (APC), such that the APCsubsequently presents the antigen to generate an immune response. Thepresent invention therefore employs a different strategy to the priorart.

STATEMENTS OF THE INVENTION

According to one aspect of the present invention there is provided adelivery system comprising a T cell comprising at least one antigen andcapable of delivering the antigen to a lymph node, or other part of thelymph system.

In other words the present invention provides a delivery systemcomprising a T cell comprising at least one antigen capable of loadingantigen presenting cells (APCs) with the antigen.

The antigen is generally foreign to the T cell. By “foreign” we includeantigens which are normally exogenous to the T cell.

The antigen may be introduced into the T cell as a polynucleotideencoding the antigen, i.e. the T cell may be transfected or transducedwith the polynucleotide, or the T cell may be loaded with the antigen inthe form of a polypeptide, but may equally well be a protein. For easeof reference, the terms polypeptide and protein and generally usedinterchangeably throughout. Thus, by the T cell comprising the antigenwe mean that the antigen is associated with the T cell such that the Tcell is capable of delivering the antigen to the lymph node and/or APCs.

According to another aspect of the present invention there is provided adelivery system comprising a T cell comprising at least one antigen andcapable of delivering the antigen to an APC for subsequent presentationby the APC.

In one embodiment the foreign antigen is a bacterial or viral antigen.Preferably the foreign antigen is a tumor antigen.

Preferably the T cell is further comprises at least one further foreignpolypeptide or polynucleotide sequence.

In one embodiment the further foreign gene is a marker, i.e. a marker orselection gene.

Preferably the marker is a bacterial resistance gene, e.g. a bacterialresistance gene which confers neomycin resistance.

In another embodiment the marker is a further antigen, preferably HSV-Tkor CD20. This antigen are particularly preferred because they can beemployed in so-called suicide systems.

In a further embodiment the delivery system further comprises both thefurther antigen and marker gene.

Preferably the T cell expresses at least one of the following markers:HLA-I, HLA-II CD80, CD86, CD27, CD40L, CD62L, CCR7, CD54 and CD25.

Thus, in another aspect of the present invention there is provided a Tcell which expresses at least one of the following markers: HLA-I,HLA-II, CD80, CD86, CD27, CD40L, CD62L, CCR7, CD54 and CD25.

According to another aspect of the present invention there is provideduse of a T cell comprising at least two antigens capable of raising animmune response, and wherein the response against one of the antigens isused for monitoring the immune response against the other antigen.

According to another aspect of the present invention there is provided amethod of monitoring the immune response to a T cell containing a firstantigen capable of raising an immune response, comprising introducing asecond antigen into the T cell.

According to another aspect of the present invention there is provided amethod of loading APCs in vivo comprising exposing the APCs to a T cellcontaining an antigen.

According to yet another aspect of the present invention there isprovided a method of obtaining a T cell for use in the method of claimcomprising

-   -   isolating a T cell;    -   activating the T cell;    -   culturing the T cell;    -   introducing an antigen into the T cell.

Preferably the T cell is transduced or transfected with a polynucleotidesequence coding for the antigen.

Preferably the T cell is activated with phytoemoagglutinine, anti-CD3monoclonal antibody or anti-CD3/CD28 monoclonal antibody-coated beads.

Preferably the T cell is cultured in the presence of growth factors,such as hu-r-IL-2.

Preferably the T cell is cultured in a culture media which comprises 5%autologous serum.

Preferably the T cell is cultured at 1×10⁶ cells/ml.

According to a further aspect of the present invention there is provideda T cell obtainable by the process of the present invention, and whichis useful as a delivery system according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Various preferred features and embodiments of the present invention willnow be described by way of non-limiting example.

Although in general the techniques mentioned herein are well known inthe art, reference may be made in particular to Sambrook et al.,Molecular Cloning, A Laboratory Manual (1989) and Ausubel et al., ShortProtocols in Molecular Biology (1999) 4^(th) Ed, John Wiley & Sons, Inc(as well as the complete version Current Protocols in MolecularBiology).

In general terms the present invention relates to in vitro manipulated Tlymphocytes (T cells) that when injected in vivo are able to carryantigens (e.g. tumor, bacterial or viral antigens) into contact withprofessional antigen presenting cells. However, the present inventionalso encompasses in vivo manipulation of T cells. More particularly, theinvention relates to: a) in vitro transduced/transfected T lymphocytescontaining exogenous genes encoding a tumor antigen, to stimulate theimmune response against the tumor; and b) T lymphocytes loaded in vitrowith peptides encoding tumor antigen epitopes. The T cells may alsocomprise a further strong antigen, e.g. HSV-Tk, to monitor vaccinationeffectiveness.

It has been reported that T cells are suitable targets for gene transferthrough, e.g. retroviral vectors. In particular the use of T cellsexpressing tumor antigens for tumor vaccination has been described.However, it has also been reported, e.g. in EP 0 905 786, that the anyeffect occurs through T cells acting as antigen presenting cells (APCs).In contrast to this view we have now found that the T cells act byexposing an APC to the transgene which is subsequently presented by theAPC. In other words, the T cells expose the transgene product to an APCfor antigen processing and presentation. By “exposing” we includebringing the transgene into contact with an APC such that binding and/oruptake of the transgene product occurs. The APC internalises the antigenand processes it for presentation.

By “antigen processing” we include the degradation of the antigen intoshorter peptide sequences and the association of the peptide with MHCmolecules. Two distinct classes of MHC molecules, MHC class I and MHCclass II, regulate the presentation of antigens to either CD8+ or CD4+ Tcells, respectively. Antigen presentation is, strictly speaking, theactivation of T cells via T cell receptors, which specifically recogniseantigenic peptide in association with either MIC class I or class IImolecules on the surface of APCs; however B cells are also capable ofrecognising and binding certain antigens.

We have also found that the present system provides an effective methodof delivering an antigen to the lymph system, and particularly the lymphnodes. The T cells may be administered locally, but we also believe thatthe T cells are capable of efficiently migrating to the lymph nodes,even if they are administered at a distal site. Lymph nodes function asan immunologic filter for the bodily fluid known as lymph. Lymph nodesare found throughout the body. Composed mostly of T cells, B cells,dendritic cells and macrophages, the nodes drain fluid from most of ourtissues. Antigens are filtered out of the lymph in the lymph node beforereturning the lymph to the circulation. In a similar fashion in thespleen, the macrophages and dendritic cells that capture antigenspresent these foreign materials to T and B cells, consequentlyinitiating an immune response.

The invention further thus provides a cell-dependent delivery system foran antigen. In this system the antigen, or a polynucleotide encoding theantigen, is introduced into one or more cells ex vivo and thenintroduced into the patient.

The T cells of the present invention may be administered alone, but willgenerally be administered as a pharmaceutical composition.

APCs and Immune Response

As mentioned above the present invention relates to delivery of anantigen to an antigen presenting cell (APC). APCs include macrophages,dendritic cells, B cells and virtually any other cell type capable ofexpressing an MHC molecule.

Macrophages are phagocytic cells of the monocytic lineage residingwithin tissues and are particularly well equipped for effective antigenpresentation. They generally express MAC class II molecules and alongwith their phagocytic properties are extremely efficient at engulfingmacromolecular or particulate material, digesting it, processing it withan extensive lysosomal system to antigenic peptide form, and expressingit on the cell surface for recognition by T lymphocytes.

Dendritic cells, so named for their highly branched morphology, arefound in many organs throughout the body, are bone marrow-derived andusually express high levels of MAC class II antigen. Dendritic cells areactively motile and can recirculate between the bloodstream and tissues.Dendritic cells are antigen-presenting-cells with a unique ability toinduce primary immune responses, thus permitting establishment ofimmunological memory. Dendritic cells in the immature state have an highphagocytic capacity. After antigen capture, immature dendritic cellsmigrate to the lymph nodes where, after maturation, activate circulatingantigen-specific lymphocytes. In this way, they too are consideredimportant APCs. Langerhans cells are an example of dendritic cells thatare located in the skin.

B lymphocytes, while not actively phagocytic, are class II-positive andpossess cell surface antigen-specific receptors, immunoglobulin, orantibody molecules. Due to their potential for high affinity antigenbinding, B cells are uniquely endowed with the capacity to concentratelow concentrations of antigen on their surface, endocytose it, processit and present it in the context of antigenic peptide in associationwith MHC antigen on their surface. In this manner, B cells becomeextremely effective APCs.

Thus, an immune response to foreign antigen requires the presence of anantigen-presenting cell (APC) (usually either a macrophage or dendriticcell), in combination with a B cell or T cell. When an APC presents anantigen on its cell surface to a B cell, the B cell is signalled toproliferate and produce antibodies that specifically bind to thatantigen. If the antibodies bind to antigens on bacteria or parasites itacts as a signal for polymorphonuclear leukocytes or macrophages toengulf (phagocytose) and kill them. Another important function ofantibodies is to initiate the “complement destruction cascade”. Whenantibodies bind to cells or bacteria, serum proteins called complementbind to the immobilized antibodies and destroy the bacteria by creatingholes in them. Antibodies can also signal natural killer cells andmacrophages to kill viral or bacterial-infected cells.

If the APC presents the antigen to T cells, the T cells becomeactivated. Activated T cells proliferate and become secretory in thecase of CD4+ T cells, or, if they are CD8+ T cells, they becomeactivated to kill target cells that specifically express the antigenpresented by the APC. The production of antibodies and the activity ofCD8+ killer T cells are highly regulated by the CD4+ helper T cellsubset. The CD4+ T cells provide growth factors or signals to thesecells that signal them to proliferate and function more efficiently.

T Cells

T cells or T lymphocytes are usually divided into two major subsets thatare functionally and phenotypically (identifiably) different. The Thelper subset, also called the CD4+ T cell, is a pertinent coordinatorof immune regulation. The main function of the T helper cell is toaugment or potentiate immune responses by the secretion of specializedfactors that activate other white blood cells to fight off infection.

Another important type of T cell is called the T killer-subset or CD8+ Tcell. These cells are important in directly killing certain tumor cells,viral-infected cells and sometimes parasites. They often depend on thesecondary lymphoid organs (the lymph nodes and spleen) as sites whereactivation occurs, but they are also found in other tissues of the body,most conspicuously the liver, lung, blood, and intestinal andreproductive tracts.

Where T cells are to be used in the ex vivo methods of the invention,the T cells are typically T lymphocytes isolated from the blood of apatient or donor. T cells are obtained by an appropriate method (e.g. asdescribed in U.S. Pat. No. 4,663,058) and may be enriched and/orpurified by standard methods including antibody-mediated separation. TheT cells may be used in combination with other immune cells, obtainedfrom the same or a different individual. Alternatively whole blood maybe used or leukocyte enriched blood or purified white blood cells as asource of T cells and other cell types. It is particularly preferred touse helper T cells (CD4⁺). Alternatively other T cells such as CD8⁺cells may be used. It may also be convenient to use cell lines such as Tcell hybridomas, immature T cells of peripheral or thymic origin andNK-T cells. In a preferred embodiment, the T cells used in the presentinvention will be T cells that can transfer antigen specific suppressionto other T cells.

Introduction of Polypeptides and Nucleic Acid Sequences into T Cells

Antigenic polypeptide substances may be administered to T cells as thepolypeptide itself or by introducing nucleic acid constructs/viralvectors encoding the polypeptide into cells under conditions that allowfor expression of the polypeptide in the T cell.

Preferably, a polynucleotide for use in the invention in a vector isoperably linked to a control sequence that is capable of providing forthe expression of the coding sequence by the host cell, i.e. the vectoris an expression vector. The term “operably linked” means that thecomponents described are in a relationship permitting them to functionin their intended manner. A regulatory sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under condition compatible with the controlsequences.

The control sequences may be modified, for example by the addition offurther transcriptional regulatory elements to make the level oftranscription directed by the control sequences more responsive totranscriptional modulators.

Vectors of the invention may be transformed or transfected into a T cellas described below to provide for expression of an antigen.

The present invention also encompasses T cells into which antigens, e.g.in the form of polypeptides, are introduced, i.e. loaded.

For ease of reference both the antigenic polypeptides andpolynucleotides encoding therefor may be referred to as “antigen”.

Convenient non-limiting methods for introducing both genes andpolypeptides into T cells are discussed below.

Any suitable method of transforming the T cell may be used. Non-limitingexamples of currently available mechanisms for delivery are viaelectroporation, calcium phosphate transformation or particlebombardment. However, transfer of the construct may be performed by anyof the methods mentioned which physically or chemically permeabilize thecell membrane. Suitable methods are described in more detail below.

1. Electroporation

In certain preferred embodiments of the present invention, the antigenis introduced into the cells via electroporation. Electroporationinvolves the exposure of a suspension of cells and DNA to a high-voltageelectric discharge.

It is contemplated that electroporation conditions for T cells may beoptimized. One may particularly with to optimize such parameters as thevoltage, the capacitance, the time and the electroporation mediacomposition. The execution of other routine adjustments will be known tothose of skill in the art.

2. Particle Bombardment

One method for transferring a naked DNA construct into cells involvesparticle bombardment. This method depends on the ability to accelerateDNA-coated microprojectiles to a high velocity allowing them to piercecell membranes and enter cells without killing them. Themicroprojectiles used have consisted of biologically inert substancessuch as tungsten, platinum or gold beads.

It is contemplated that in some instances DNA precipitation onto metalparticles would not be necessary for DNA delivery to a recipient cellusing particle bombardment. It is contemplated that particles maycontain DNA rather than be coated with DNA. Hence it is proposed thatDNA-coated particles may increase the level of DNA delivery via particlebombardment but are not, in and of themselves, necessary.

Several devices for accelerating small particles have been developed.One such device relies on a high voltage discharge to generate anelectrical current, which in turn provides the motive force. Anothermethod involves the use of a Biolistic Particle Delivery System, whichcan be used to propel particles coated with DNA through a screen, suchas stainless steel or Nytex screen, onto a filter surface covered withcells in suspension. The screen disperses the particles so that they arenot delivered to the recipient cells in large aggregates. It is believedthat a screen intervening between the projectile apparatus and the cellsto be bombarded reduces the size of projectile aggregates and maycontribute to a higher frequency of transformation by reducing thedamage inflicted on the recipient cells by projectiles that are toolarge.

For the bombardment, cells in suspension are preferably concentrated onfilters, or alternatively on solid culture medium. The cells to bebombarded are positioned at an appropriate distance below themacroprojectile stopping plate. If desired, one or more screens are alsopositioned between the acceleration device and the cells to bebombarded.

It is contemplated that one may wish to adjust various of thebombardment parameters in small scale studies to fully optimize theconditions. One may particularly wish to adjust physical parameters suchas gap distance, flight distance, tissue distance and helium pressure.One may also optimize the trauma reduction factors by modifyingconditions which influence the physiological state of the recipientcells and which may therefore influence transformation and integrationefficiencies. For example, the osmotic state, tissue hydration and thesubculture stage or cell cycle of the recipient T cells may be adjustedfor optimum transformation. The execution of other routine adjustmentswill be known to those of skill in the art.

3. Viral Transformation

a. Adenoviral Infection

One method for delivery of the nucleic acid constructs involves the useof an adenovirus expression vector. Although adenovirus vectors areknown to have a low capacity for integration into genomic DNA, thisfeature is counterbalanced by the high efficiency of gene transferafforded by these vectors. “Adenovirus expression vector” is meant toinclude those constructs containing adenovirus sequences sufficient to(a) support packaging of the construct and (b) to ultimately express aconstruct that has been cloned therein.

The vector comprises a genetically engineered form of adenovirus.Knowledge of the genetic organization or adenovirus, a 36 kb, linear,double-stranded DNA virus, allows substitution of large pieces ofadenoviral DNA with foreign sequences up to 7 kb. In contrast toretrovirus, the adenoviral infection of host cells does not result inchromosomal integration because adenoviral DNA can replicate in anepisomal manner without potential genotoxicity. Also, adenoviruses arestructurally stable, and no genome rearrangement has been detected afterextensive amplification.

Adenovirus is particularly suitable for use as a gene transfer vectorbecause of its mid-sized genome, ease of manipulation, high titer, widetarget-cell range and high infectivity. Both ends of the viral genomecontain 100-200 base pair inverted repeats (ITRs), which are ciselements necessary for viral DNA replication and packaging. The early(E) and late (L) regions of the genome contain different transcriptionunits that are divided by the onset of viral DNA replication. The E1region (E1A and E1B) encodes proteins responsible for the regulation oftranscription of the viral genome and a few cellular genes. Theexpression of the E2 region (E2A and E2B) results in the synthesis ofthe proteins for viral DNA replication. These proteins are involved inDNA replication, late gene expression and host cell shut-off. Theproducts of the late genes, including the majority of the viral capsidproteins, are expressed only after significant processing of a singleprimary transcript issued by the major late promoter (MLP). The MLP,(located at 16.8 m.u.) is particularly efficient during the late phaseof infection, and all the mRNA's issued from this promoter possess a5′-tripartite leader (TPL) sequence which makes them preferred mRNA'sfor translation.

In a current system, recombinant adenovirus is generated from homologousrecombination between shuttle vector and provirus vector. Due to thepossible recombination between two proviral vectors, wild-typeadenovirus may be generated from this process. Therefore, it is criticalto isolate a single clone of virus from an individual plaque and examineits genomic structure.

Generation and propagation of the current adenovirus vectors, which arereplication deficient, depend on a unique helper cell line, designated293, which was transformed from human embryonic kidney cells by Ad5 DNAfragments and constitutively expresses E1 proteins. Since the E3 regionis dispensable from the adenovirus genome the current adenovirusvectors, with the help of 293 cells, carry foreign DNA in either the E1,the D3 or both regions. In nature, adenovirus can package approximately105% of the wild-type genome, providing capacity for about 2 extra kb ofDNA. Combined with the approximately 5.5 kb of DNA that is replaceablein the E1 and E3 regions, the maximum capacity of the current adenovirusvector is under 7.5 kb, or about 15% of the total length of the vector.More than 80% of the adenovirus viral genome remains in the vectorbackbone.

Helper cell lines may be derived from human cells such as humanembryonic kidney cells, muscle cells, hematopoietic cells or other humanembryonic mesenchymal or epithelial cells. Alternatively, the helpercells may be derived from the cells of other mammalian species that arepermissive for human adenovirus. Such cells include, e.g., Vero cells orother monkey embryonic mesenchymal or epithelial cells. As stated above,the preferred helper cell line is 293.

Other than the requirement that the adenovirus vector be replicationdefective, or at least conditionally defective, the nature of theadenovirus vector is not believed to be crucial to the successfulpractice of the invention. The adenovirus may be of any of the 42different known serotypes or subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain theconditional replication-defective adenovirus vector for use in thepresent invention. This is because Adenovirus type 5 is a humanadenovirus about which a great deal of biochemical and geneticinformation is known, and it has historically been used for mostconstructions employing adenovirus as a vector.

As stated above, the typical vector according to the present inventionis replication defective and will not have an adenovirus E1 region.Thus, it will be most convenient to introduce the transforming constructat the position from which the E1-coding sequences have been removed.However, the position of insertion of the construct within theadenovirus sequences is not critical to the invention. Thepolynucleotide encoding the gene of interest may also be inserted inlieu of the deleted E3 region in E3 replacement vectors or in the E4region where a helper cell line or helper virus complements the E4defect.

Adenovirus growth and manipulation is known to those of skill in theart, and exhibits broad host range in vitro and in vivo. T is group ofviruses can be obtained in high titers, e.g., 10.sup.9-10.sup.11plaque-forming units per ml, and they are highly infective. The lifecycle of adenovirus does not require integration into the host cellgenome. The foreign genes delivered by adenovirus vectors are episomaland, therefore, have low genotoxicity to host cells. No side effectshave been reported in studies of vaccination with wild-type adenovirus,demonstrating their safety and therapeutic potential also as in vivogene transfer vectors.

b. AAV Infection

Adeno-associated virus (AAV) is an attractive vector system for use inthe present invention as it has a high frequency of integration and itcan infect nondividing cells, thus making it useful for delivery ofgenes into mammalian cells in tissue culture. AAV has a broad host rangefor infectivity. Details concerning the generation and use of rAAVvectors are described in U.S. Pat. No. 5,139,941 and U.S. Pat. No.4,797,368, each incorporated herein by reference.

Recombinant AAV vectors have been used successfully for in vitro and invivo transduction of marker genes and genes involved in human diseases.Recently, an AAV vector has been approved for phase I human trials forthe treatment of cystic fibrosis.

AAV is a dependent parvovirus in that it requires coinfection withanother virus (either adenovirus or a member of the herpes virus family)to undergo a productive infection in cultured cells. In the absence ofcoinfection with helper virus, the wild type AAV genome integratesthrough its ends into human chromosome 19 where it resides in a latentstate as a provirus. rAAV, however, is not restricted to chromosome 19for integration unless the AAV Rep protein is also expressed. When acell carrying an AAV provirus is superinfected with a helper virus, theAAV genome is “rescued” from the chromosome or from a recombinantplasmid, and a normal productive infection is established.

Typically, recombinant AAV (rAAV) virus is made by cotransfecting aplasmid containing the gene of interest flanked by the two AAV terminalrepeats and an expression plasmid containing the wild type AAV codingsequences without the terminal repeats, for example pIM45. The cells arealso infected or transfected with adenovirus or plasmids carrying theadenovirus genes required for AAV helper function. rAAV virus stocksmade in such fashion are contaminated with adenovirus which must bephysically separated from the rAAV particles (for example, by cesiumchloride density centrifugation). Alternatively, adenovirus vectorscontaining the AAV coding regions or cell lines containing the AAVcoding regions and some or all of the adenovirus helper genes could beused. Cell lines carrying the rAAV DNA as an integrated provirus canalso be used.

c. Retroviral Infection

One preferred method of the present invention involves the use ofretroviruses. The retroviral vector of the present invention may bederived from or may be derivable from any suitable retrovirus. A largenumber of different retroviruses have been identified. Examples include:murine leukemia virus (MLV), human immunodeficiency virus (HIV), simianimmunodeficiency virus, human T-cell leukemia virus (HTLV). equineinfectious anaemia virus (EIAV), mouse mammary tumor virus (MMTV), Roussarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murineleukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV),Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus(A-MLV), Avian myelocytomatosis virus-29 (MC29), and Aviancrythroblastosis virus (AEV). A detailed list of retroviruses may befound in Coffin et al., 1997, “retroviruses”, Cold Spring HarbourLaboratory Press Eds: J M Coffin, S M Hughes, H E Varmus pp 758-763.

Details on the genomic structure of some retroviruses may be found inthe art. By way of example, details on HIV and Mo-MLV may be found fromthe NCBI Genbank (Genome Accession Nos. AF033819 and AF033811,respectively).

Retroviruses may be broadly divided into two categories: namely,“simple” and “complex”. Retroviruses may even be further divided intoseven groups. Five of these groups represent retroviruses with oncogenicpotential. The remaining two groups are the lentiviruses and thespumaviruses. A review of these retroviruses is presented in Coffin etal., 1997 (ibid).

The lentivirus group can be split even further into “primate” and“on-primate”. Examples of primate lentiviruses include humanimmunodeficiency virus (HIV), the causative agent of humanauto-immunodeficiency syndrome (AIDS), and simian immunodeficiency virus(SIV). The non-primate lentiviral group includes the prototype “slowvirus” visna/maedi virus (VMV), as well as the related caprinearthritis-encephalitis virus (CAEV), equine infectious anaemia virus(EIAV) and the more recently described feline immunodeficiency virus(FIV) and bovine immunodeficiency virus (BIV).

A distinction between the lentivirus family and other types ofretroviruses is that lentiviruses have the capability to infect bothdividing and non-dividing cells. In contrast, other retroviruses—such asMLV—are unable to infect non-dividing cells such as those that make up,for example, muscle, brain, lung and liver tissue.

Each retroviral genome comprises genes called gag, pot and env whichcode for virion proteins and enzymes. In the provirus, these genes areflanked at both ends by regions called long terminal repeats (LTRs). TheLTRs are responsible for proviral integration, and transcription. LTRsalso serve as enhancer-promoter sequences and can control the expressionof the viral genes. Encapsidation of the retroviral RNAs occurs byvirtue of a psi sequence located at the 5′ end of the viral genome.

The LTRs themselves are identical sequences that can be divided intothree elements, which are called U3, R and U5. U3 is derived from thesequence unique to the 3′ end of the RNA. R is derived from a sequencerepeated at both ends of the RNA and U5 is derived from the sequenceunique to the 5′ end of the RNA. The sizes of the three elements canvary considerably among different retroviruses.

The basic molecular organisation of an infectious retroviral RNA genomeis (5′) R-U5-gag, pol, env-U3-R (3′). In a defective retroviral vectorgenome gag, pol and env may be absent or not functional. The R regionsat both ends of the RNA are repeated sequences. U5 and U3 representunique sequences at the 5′ and 3′ ends of the RNA genome respectively.

In order to construct a retroviral vector, a nucleic acid encoding agene of interest is inserted into the viral genome in the place ofcertain viral sequences to produce a virus that isreplication-defective. In order to produce virions, a packaging cellline containing the gag, pol, and env genes but without the LTR andpackaging components is constructed (Mann et al., 1983). When arecombinant plasmid containing a cDNA, together with the retroviral LTRand packaging sequences is introduced into this cell line (by calciumphosphate precipitation for example), the packaging sequence allows theRNA transcript of the recombinant plasmid to be packaged into viralparticles, which are then secreted into the culture media. The mediacontaining the recombinant retroviruses is then collected, optionallyconcentrated, and used for gene transfer. Retroviral vectors are able toinfect a broad variety of cell types. However, integration and stableexpression require the division of host cells.

Concern with the use of defective retrovirus vectors is the potentialappearance of wild-type replication-competent virus in the packagingcells. This can result from recombination events in which the intactsequence from the recombinant virus inserts upstream from the gag, pol,env sequence integrated in the host cell genome. However, new packagingcell lines are now available that should greatly decrease the likelihoodof recombination.

d. Other Viral Vectors

Other viral vectors may be employed as constructs in the methods andcompositions described here. Vectors derived from viruses such asvaccinia and herpesviruses may be employed.

4. Calcium Phosphate Co-Precipitation or DEAE-Dextran Treatment

In other preferred embodiments, antigen is introduced to the cells usingcalcium phosphate co-precipitation. In another embodiment, theexpression construct is delivered into the cell using DEAE-dextranfollowed by polyethylene glycol.

5. Direct Microinjection or Sonication Loading

Further embodiments include the introduction of the antigen by directmicroinjection or sonication loading.

6. Liposome Mediated Transformation

In a further embodiment, the antigen may be entrapped in a liposome.Liposomes are vesicular structures characterized by a phospholipidbilayer membrane and an inner aqueous medium. Multilamellar liposomeshave multiple lipid layers separated by aqueous medium. They formspontaneously when phospholipids are suspended in an excess of aqueoussolution. The lipid components undergo self-rearrangement before theformation of closed structures and entrap water and dissolved solutesbetween the lipid bilayers. Also contemplated is a nucleic acidconstruct complexed with Lipofectamine (Gibco BRL).

In certain embodiments of the invention, the liposome may be complexedwith a hemagglutinating virus (HVJ). This has been shown to facilitatefusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA. In other embodiments, the liposome may becomplexed or employed in conjunction with nuclear non-histonechromosomal proteins (HMG-1). In yet further embodiments, the liposomemay be complexed or employed in conjunction with both HVJ and HMG-1.

7. Adenoviral Assisted Transfection

In certain embodiments, the nucleic acid construct is introduced intothe cell using adenovirus assisted transfection. Increased transfectionefficiencies have been reported in cell systems using adenovirus coupledsystems, and the inventors contemplate using the same technique toincrease transfection efficiencies.

Culturing

The present invention also provides a method of culturing T cells foruse in the present invention.

In particular, peripheral blood mononuclear cells (PBMC) isolated fromperipheral blood may be activated in vitro to proliferate Mitogenicstimulation may be obtained by the use of compounds such asphytoemoagglutinine (PHA; 2 μg/ml) or anti-CD3 mAb (OKT3; 30 ng/ml).

Activated T cells may be cultured at 1×10⁶ cells/ml, in mediasupplemented with 5% autologous serum in the presence of growth factors,such as hu-r-IL-2 (e.g. 100 U/ml).

One preferred embodiment the T cells are cultured in the presence ofmagnetic beads coated with antibodies to CD3 and CD28, preferably in thepresence of hu-r-IL2 (e.g. 100 U/ml). Conveniently the beads are used ata ratio of three beads/T cell.

The T cells may then be transduced/transfected by exposure of theactivated T cells e.g. using viral vector(s) encoding the antigen(s).The expression of the transgene(s) may be used to evaluate transductionefficiency. Selection genes and markers is discussed in more detailbelow. Alternatively, lymphocytes may cultured as described above, butnot transduced/transfected. Instead, before administration to a patient,activated T cells are loaded with antigen, e.g. in the form of syntheticpeptides encoding e.g. well-known tumor, bacterial or viral antigens

The entire manipulation process generally lasts 5-15 days.

The manipulated T cells may be frozen for storage purposes.

At the end of the culture the manipulated T cells should express atleast one of the following markers HLA-I HLA-II CD80, CD86, CD27, CD40LCD62L CCR7, CD54 and CD25. Thus, we have found that the phenotype of theT cell is important.

Vaccine

The invention relates to a method for inducing an immunological responsein an individual, particularly a mammal, preferably humans, whichcomprises inoculating the individual with the antigen-containing T cellof the present invention, such that the antigen produces an antibodyand/or T cell immune response to protect said individual from, forexample, a tumor or infection, such as a bacterial or viral infection.Also provided are methods whereby such immunological response slowstumor growth or viral or bacterial replication.

Thus, the invention relates to an immunological composition that whenintroduced into an individual, preferably a human, capable of havinginduced within it an immunological response, induces an immunologicalresponse in such individual. The immunological response may be usedtherapeutically or prophylactically and may take the form of antibodyimmunity and/or cellular immunity, such as cellular immunity arisingfrom CTL or CD4+ T cells.

The preparation of vaccines which contain an immunogenic polypeptide(s)as active ingredient(s), is known to one skilled in the art. Typically,such vaccines are prepared as injectables, either as liquid solutions orsuspensions; solid forms suitable for solution in, or suspension in,liquid prior to injection may also be prepared. The preparation may alsobe emulsified, or the protein encapsulated in liposomes. The activeimmunogenic ingredients are often mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredient.Suitable excipients are, for example, water, saline, dextrose, glycerol,ethanol, or the like and combinations thereof.

In addition, if desired, the vaccine may contain minor amounts ofauxiliary substances such as wetting or emulsifying agents, pH bufferingagents, and/or adjuvants which enhance the effectiveness of the vaccine.

The vaccine formulation of the invention preferably relates to and/orincludes an adjuvant system for enhancing the immunogenicity of theformulation. Preferably the adjuvant system raises preferentially a TH1type of response.

An immune response may be broadly distinguished into two extremecategories, being a humoral or cell mediated immune responses(traditionally characterised by antibody and cellular effectormechanisms of protection respectively). These categories of responsehave been termed TH1-type responses (cell-mediated response), andTH2-type immune responses (humoral response).

Extreme TH1-type immune responses may be characterised by the generationof antigen specific, haplotype restricted cytotoxic T lymphocytes, andnatural killer cell responses. In mice TH1-type responses are oftencharacterised by the generation of antibodies of the IgG2a subtype,whilst in the human these correspond to IgG1 type antibodies. TH2-typeimmune responses are characterised by the generation of a broad range ofimmunoglobulin isotypes including in mice IgG1, IgA, and IgM.

It can be considered that the driving force behind the development ofthese two types of immune responses are cytokines. High levels ofTH1-type cytokines tend to favour the induction of cell mediated immuneresponses to the given antigen, whilst high levels of TH2-type cytokinestend to favour the induction of humoral immune responses to the antigen.

Immunomodulators, such as vaccines may be prepared from T cellscomprising one or more polypeptides, or even nucleotide sequences, ofthe present invention. The preparation of immunomodulators which containan immunogenic polypeptide(s) as active ingredient(s), is known to oneskilled in the art. Typically, such immunomodulators are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectionmay also be prepared. The preparation may also be emulsified, or theprotein encapsulated in liposomes. The active immunogenic ingredientsare often mixed with excipients which are pharmaceutically acceptableand compatible with the active ingredient. Suitable excipients are, forexample, water, saline, dextrose, glycerol, ethanol, or the like andcombinations thereof.

In addition, if desired, the immunomodulators may contain minor amountsof auxiliary agents such as wetting or emulsifying agents, pH bufferingagents, and/or adjuvants which enhance the effectiveness of theimmunomodulators. Examples of adjuvants which may be effective includebut are not limited to: aluminum hydroxide,N-acetyl-muramyl-L-threonyl-D-isoglutamine (tbr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE), and RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween80 emulsion.

Further examples of adjuvants and other agents include aluminumhydroxide, aluminum phosphate, aluminum potassium sulfate (alum),beryllium sulfate, silica, kaolin, carbon, water-in-oil emulsions,oil-in-water emulsions, muramyl dipeptide, bacterial endotoxin, lipid X,Corynebacterium parvum (Propionobacterium acnes), Bordetella pertussis,polyribonucleotides, sodium alginate, lanolin, lysolecithin, vitamin A,saponin, liposomes, levamisole, DEAE-dextran, blocked copolymers orother synthetic adjuvants. Such adjuvants are available commerciallyfrom various sources, for example, Merck Adjuvant 65 (Merck and Company,Inc., Rahway, N.J.) or Freund's Incomplete Adjuvant and CompleteAdjuvant (Difco Laboratories, Detroit, Mich.).

Typically, adjuvants such as Amphigen (oil-in-water), Alhydrogel(aluminum hydroxide), or a mixture of Amphigen and Alhydrogel are used.Only aluminum hydroxide is approved for human use.

The proportion of immunogen and adjuvant can be varied over a broadrange so long as both are present in effective amounts. For example,aluminum hydroxide can be present in an mount of about 0.5% of theimmunomodulators mixture (Al₂O₃ basis). Conveniently, theimmunomodulators are formulated to contain a final concentration ofimmunogen in the range of from 0.2 to 200 μg/ml, preferably 5 to 50μg/ml, most preferably 15 μg/ml.

After formulation, the immunomodulators may be incorporated into asterile container which is then sealed and stored at a low temperature,for example 4° C., or it may be freeze-dried. Lyophilisation permitslong-term storage in a stabilised form.

The immunomodulators are conventionally administered parenterally, byinjection, for example, either subcutaneously or intramuscularly.Additional formulations which are suitable for other modes ofadministration include suppositories and, in some cases, oralformulations. For suppositories, traditional binders and carriers mayinclude, for example, polyalkylene glycols or triglycerides; suchsuppositories may be formed from mixtures containing the activeingredient in the range of 0.5% to 10%, preferably 1% to 2%. Oralformulations include such normally employed excipients as, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, and the like. Thesecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders and contain 10% to95% of active ingredient, preferably 25% to 70%. Where theimmunomodulators composition is lyophilised, the lyophilised materialmay be reconstituted prior to administration, e.g. as a suspension.Reconstitution is preferably effected in buffer.

The polypeptides of the invention may be formulated into theimmunomodulators as neutral or salt forms. Pharmaceutically acceptablesalts include the acid addition salts (formed with free amino groups ofthe peptide) and which are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such organic acids such asacetic, oxalic, tartaric and maleic. Salts formed with the free carboxylgroups may also be derived from inorganic bases such as, for example,sodium, potassium, ammonium, calcium, or ferric hydroxides, and suchorganic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol,histidine and procaine.

Antigen

In general terms an antigen is any substance that is capable ofinteracting with an antigen receptor. An antigen binds to a specificantibody to provide a mechanism by which the antigen is recognised andinactivated, in this manner, an antigen complexes with a specificantibody so that the complex can attach itself to specialised immunecells that either internalise the complex to destroy it or releasebiological mediators such as histamine to produce anallergic/inflammatory response. An immunogen is an antigen thatactivates immune cells to generate an immune response against itself.Thus, an immunogen is an antigen, but an antigen is not necessarily animmunogen. However, for ease of reference herein the terms antigen andimmunogen are generally used interchangeably.

The present invention in general terms relates to a new method ofpresenting an antigen to the immune system. Some of the factors thatdetermine the immunogenic potential of an antigen, and hence its abilityto generate an immune response, include accessibility of an antigenicepitope to immune cell recognition and the route of antigenadministration. The present invention provides a system for improvingthese factors such that the immunogenicity of an antigen is optimised.As a result, someone may be able to receive the equivalent of say two orthree doses in just one administration of vaccine. Thus, not only is theefficacy of the vaccine improved, but also patient compliance.

The present invention is applicable to any antigen which is capable ofbeing introduced into a T cell such that the antigen is presented to theimmune system, and more particularly an APC. In a particularly preferredembodiment the antigen is introduced into the T cell in the form of apolynucleotide which is capable of being expressed in the T cell.

A wide range of suitable antigens is known. Their sequence may beselected, for example, on the basis of polypeptide sequences known inthe literature. For polypeptides which have a partial sequence of aprotein with an immunogenic activity, it is possible to establish whichpeptides are suitable candidates by means of sequence comparison.Preferred candidates are polypeptides whose immunogenicity has alreadybeen demonstrated, i.e. polypeptides derived from known immunogens, suchas viral or bacterial proteins. Polypeptides of this type preferablyexhibit a strong reaction on an MLC test on account of theirimmunogenicity. The term “protein” includes single-chain polypeptidemolecules as well as multiple-polypeptide complexes where individualconstituent polypeptides are linked by covalent or non-covalent means.The term “polypeptide” includes peptides of two or more amino acids inlength, typically having more than 5, 10 or 20 amino acids.

It will be understood that polypeptide sequences for use in theinvention are not limited to particular sequences or fragments thereofor sequences obtained from a particular protein but also includehomologous sequences obtained from any source, for example relatedviral/bacterial proteins, cellular homologues and synthetic peptides, aswell as variants or derivatives thereof. Polypeptide sequences of thepresent invention also include polypeptides encoded by polynucleotidesof the present invention.

Thus, the present invention encompasses variants, homologues orderivatives of the amino acid sequences of antigens, as well asvariants, homologues or derivatives of the amino acid sequences codedfor by the nucleotide sequences of the present invention.

In the context of the present invention, a homologous sequence is takento include an amino acid sequence which is at least 60, 70, 80 or 90%identical, preferably at least 95 or 98% identical at the amino acidlevel with a particular sequence. In particular, homology shouldtypically be considered with respect to those regions of the sequenceknown to be essential for antigenicity rather than non-essentialneighbouring sequences. Regions that are conserved across family membersshould be required to have relatively high homology scores—this assistsin defining functional molecules. Regions that are unique or poorlyconserved between family members should/may be allowed to have lowerscore, on a case by case basis—this assists in establishing novelty.This bit by bit approach is in general more useful than an overallhomology score. Although homology can also be considered in terms ofsimilarity (i.e. amino acid residues having similar chemicalproperties/functions), in the context of the present invention it ispreferred to express homology in terms of sequence identity.

Homology comparisons can be conducted by eye, or more usually, with theaid of readily available sequence comparison programs. Thesecommercially available computer programs can calculate % homologybetween two or more sequences.

The terms “variant” or “derivative” in relation to the amino acidsequences of the present invention includes any substitution of,variation of, modification of, replacement of, deletion of or additionof one (or more) amino acids from or to the sequence providing theresultant amino acid sequence preferably has antigenic activity,preferably having at least 25 to 50% of the activity as the polypeptidespresented in the sequence listings, more preferably at leastsubstantially the same activity.

Thus, particular sequences of the invention may be modified for use inthe present invention. Typically, modifications are made that maintainthe antigenicity of the sequence. Thus, in one embodiment, amino acidsubstitutions may be made, for example from 1, 2 or 3 to 10, 20 or 30substitutions provided that the modified sequence retains at least about25 to 50% of, or substantially the same activity. However, in analternative embodiment, modifications to the amino acid sequences of apolypeptide of the invention may be made intentionally to reduce thebiological activity of the polypeptide. For example truncatedpolypeptides that remain capable of binding to target molecule but lackfunctional effector domains may be useful as inhibitors of thebiological activity of the full length molecule.

In general, preferably less than 20%, 10% or 5% of the amino acidresidues of a variant or derivative are altered as compared with thecorresponding region depicted in the sequence listings.

Amino acid substitutions may include the use of non-naturally occurringanalogues, for example to increase blood plasma half-life of atherapeutically administered polypeptide (see below for further detailson the production of peptide derivatives for use in therapy).

Conservative substitutions may be made, for example according to theTable below. Amino acids in the same block in the second column andpreferably in the same line in the third column may be substituted foreach other:

ALIPHATIC Non-polar G A P I L V Polar - uncharged C S T M N Q Polar -charged D E K R AROMATIC H F W Y

Polypeptides of the invention also include fragments of full lengthpolypeptides and variants thereof. Preferred fragments include thosewhich include an epitope. Suitable fragments will be at least about 5,e.g. 10, 12, 15 or 20 amino acids in length. They may also be less than200, 100 or 50 amino acids in length. Polypeptide fragments of theproteins and allelic and species variants thereof may contain one ormore (e.g. 2, 3, 5, or 10) substitutions, deletions or insertions,including conserved substitutions. Where substitutions, deletion and/orinsertions have been made, for example by means of recombinanttechnology, preferably less than 20%, 10% or 5% of the amino acidresidues depicted in the sequence listings are altered.

Particularly preferred fragments include those having antigenic domains.

Polynucleotides for use in the invention comprise nucleic acid sequencesencoding the sequences of the invention. It will be understood by askilled person that numerous different polynucleotides can encode thesame polypeptide as a result of the degeneracy of the genetic code. Inaddition, it is to be understood that skilled persons may, using routinetechniques, make nucleotide substitutions that do not affect thepolypeptide sequence encoded by the polynucleotides of the invention toreflect the codon usage of any particular host organism in which thepolypeptides of the invention are to be expressed.

Polynucleotides of the invention may comprise DNA or RNA. They may besingle-stranded or double-stranded. They may also be polynucleotideswhich include within them synthetic or modified nucleotides. A number ofdifferent types of modification to oligonucleotides are known in theart. These include methylphosphonate and phosphorothioate backbones,addition of acridine or polylysine chains at the 3′ and/or 5′ ends ofthe molecule. For the purposes of the present invention, it is to beunderstood that the polynucleotides described herein may be modified byany method available in the art. Such modifications may be carried outin order to enhance the in vivo activity or life span of polynucleotidesof the invention.

The terms “variant”, “homologue” or “derivative” in relation to thenucleotide sequence include any substitution of, variation of,modification of, replacement of, deletion of or addition of one (ormore) nucleic acid from or to the sequence providing the resultantnucleotide sequence codes for a polypeptide having antigenic activity.

As indicated above, with respect to sequence homology, preferably thereis at least 75%, more preferably at least 85%, more preferably at least90% homology to the sequences shown in the sequence listing herein. Morepreferably there is at least 95%, more preferably at least 98%,homology. Nucleotide homology comparisons may be conducted as describedabove. A preferred sequence comparison program is the GCG WisconsinBestfit program described above. The default scoring matrix has a matchvalue of 10 for each identical nucleotide and −9 for each mismatch. Thedefault gap creation penalty is −50 and the default gap extensionpenalty is −3 for each nucleotide.

The present invention also encompasses nucleotide sequences that arecapable of hybridising selectively to the antigenic sequences, or anyvariant, fragment or derivative thereof, or to the complement of any ofthe above. Nucleotide sequences are preferably at least 15 nucleotidesin length, more preferably at least 20, 30, 40 or 50 nucleotides inlength.

The term “hybridization” as used herein shall include “the process bywhich a strand of nucleic acid joins with a complementary strand throughbase pairing” as well as the process of amplification as carried out inpolymerase chain reaction technologies.

Polynucleotides of the invention capable of selectively hybridising tothe nucleotide sequences presented herein, or to their complement, willbe generally at least 70%, preferably at least 80 or 90% and morepreferably at least 95% or 98% homologous to the correspondingnucleotide sequences over a region of at least 20, preferably at least25 or 30, for instance at least 40, 60 or 100 or more contiguousnucleotides.

The term “selectively hybridizable” means that the polynucleotide usedas a probe is used under conditions where a target polynucleotide of theinvention is found to hybridize to the probe at a level significantlyabove background. The background hybridization may occur because ofother polynucleotides present, for example, in the cDNA or genomic DNAlibrary being screening. In this event, background implies a level ofsignal generated by interaction between the probe and a non-specific DNAmember of the library which is less than 10 fold, preferably less than100 fold as intense as the specific interaction observed with the targetDNA. The intensity of interaction may be measured, for example, byradiolabelling the probe, e.g. with ³²P.

Hybridization conditions are based on the melting temperature (Tm) ofthe nucleic acid binding complex, as taught in Berger and Kimmel (1987,Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152,Academic Press, San Diego Calif.), and confer a defined “stringency” asexplained below.

Maximum stringency typically occurs at about Tm-5° C. (5° C. below theTm of the probe); high stringency at about 5° C. to 10° C. below Tm;intermediate stringency at about 10° C. to 20° C. below Tm; and lowstringency at about 20° C. to 25° C. below Tm. As will be understood bythose of skill in the art, a maximum stringency hybridization can beused to identify or detect identical polynucleotide sequences while anintermediate (or low) stringency hybridization can be used to identifyor detect similar or related polynucleotide sequences.

In a preferred aspect, the present invention covers nucleotide sequencesthat can hybridise to an antigenic nucleotide sequence under stringentconditions (e.g. 65° C. and 0.1×SSC {1×SSC=0.15 M NaCl, 0.015 M Na₃Citrate pH 7.0}).

Where the polynucleotide of the invention is double-stranded, bothstrands of the duplex, either individually or in combination, areencompassed by the present invention. Where the polynucleotide issingle-stranded, it is to be understood that the complementary sequenceof that polynucleotide is also included within the scope of the presentinvention.

Polynucleotides which are not 100% homologous to the sequences of thepresent invention but fall within the scope of the invention can beobtained in a number of ways. Other variants of the sequences describedherein may be obtained for example by probing DNA libraries made from arange of individuals, for example individuals from differentpopulations. In addition, other viral/bacterial, or cellular homologuesparticularly cellular homologues found in mammalian cells (e.g. rat,mouse, bovine and primate cells), may be obtained and such homologuesand fragments thereof in general will be capable of selectivelyhybridising to the sequences shown in the sequence listing herein. Suchsequences may be obtained by probing cDNA libraries made from or genomicDNA libraries from other animal species, and probing such libraries withprobes comprising all or part of known sequences under conditions ofmedium to high stringency. Similar considerations apply to obtainingspecies homologues and allelic variants.

Variants and strain/species homologues may also be obtained usingdegenerate PCR which will use primers designed to target sequenceswithin the variants and homologues encoding conserved amino acidsequences within the sequences of the present invention. Conservedsequences can be predicted, for example, by aligning the amino acidsequences from several variants/homologues. Sequence alignments can beperformed using computer software known in the art. For example the GCGWisconsin PileUp program is widely used.

The primers used in degenerate PCR will contain one or more degeneratepositions and will be used at stringency conditions lower than thoseused for cloning sequences with single sequence primers against knownsequences.

Alternatively, such polynucleotides may be obtained by site-directedmutagenesis of characterised sequences. This may be useful where forexample silent codon changes are required to sequences to optimise codonpreferences for a particular host cell in which the polynucleotidesequences are being expressed. Other sequence changes may be desired inorder to introduce restriction enzyme recognition sites, or to alter theproperty or function of the polypeptides encoded by the polynucleotides.

Polynucleotides such as a DNA polynucleotides and probes according tothe invention may be produced recombinantly, synthetically, or by anymeans available to those of skill in the art. They may also be cloned bystandard techniques.

When proteins or protein fragments are used, the identity of theprocessed end product can be demonstrated by chemical analysis or bybiological assays (the ability of APCs to stimulate T-cells which arespecific to the processed fragments).

In principle, peptide candidates are selected for their suitability asforeign peptides in several stages; generally, the candidates are firsttested in a peptide binding test for their binding capacity to an MHC-Imolecule, preferably by series of tests.

One suitable method of investigation is, for example, the FACS analysisbased on flow cytometry. The peptide is marked with a fluorescent dye,e.g. with FITC (fluorescein isothiocyanate) and applied to tumor cellswhich express the MHC-I molecule. In the flow, individual cells areexcited by a laser of a certain wavelength; the fluorescence emitted ismeasured and is dependent on the quantity of peptide bound to the cell.

Another method of determining the quantity of peptide bound is theScatchard blot. Peptide labelled with I¹²⁵ or with rare earth metal ions(e.g. europium) is used for this. The cells are charged at 4° C. withvarious defined concentrations of peptide for 30 to 240 minutes. Inorder to determine non-specific interaction of peptide with cells, anexcess of unlabelled peptide is added to some of the samples, preventingthe specific interaction of the labelled peptide. Then the cells arewashed to remove any non-specific cell-associated material. The quantityof cell-bound peptide is then determined either in a scintillationcounter using the radioactivity emitted, or in a photometer which issuitable for measuring long-lived fluorescence. The data thus obtainedare evaluated using standard methods.

In a second step, candidates with good binding qualities are tested fortheir immunogenicity.

The immunogenicity of xenopeptides derived from proteins the immunogenicactivity of which is unknown may be tested, for example, by the MLCtest. Peptides which provoke a particularly violent reaction in thistest, which is preferably also carried out in series with differentpeptides, using as standard a peptide with a known immunogenic activity,are suitable for the purposes of the present invention.

Another possible way of testing MHC-I-binding peptide candidates fortheir immunogenicity consists in investigating the binding of thepeptides to T2 cells. One such test is based on the peculiar nature ofT2 cells or AMA-S-cells that they are defective in the TAP peptidetransporting mechanism and only present stable MHC-I molecules when theyare applied to peptides which are presented in the MHC-I context.

T2 cells or RMA-S cells stably transfected with an HLA gene, e.g. withHLA-A1 and/or HLA-A2 genes, are used for this test. If the cells areacted upon by peptides which are good MHC-I ligands, by being presentedin the MHC-I context in such a way as to be recognised as foreign by theimmune system, these peptides cause the HLA molecules to appear insignificant quantities on the cell surface. Detection of the HLAs on thecell surface, e.g. by means of monoclonal antibodies, makes it possibleto identify suitable peptides. Here again, a standard peptide known tohave a good HLA- or MHC-binding capacity is appropriately used.

The antigen may be derived from fungi, parasites, bacteria and viruses.Thus, one embodiment of the invention relates to the prevention ortreatment of infectious diseases. Examples of infectious diseases towhich the present invention may applied include: adenovirus, anthrax,cholera, diphtheria, tetanus, pertussis, malaria, influenza includingHaemophilus influenzae type B, Hepatitis A, Hepatitis B, encephalitis,measles, mumps, rubella, meningitis such as Meningoccal types A, C, Yand W-135, plague, pneumococcus, rabies, smallpox, salmonella, typhoid,varicella, yellow fever, Rift Valley fever. However, the presentinvention may also be applicable to use with any new immunogen whichbecomes available. Indeed, because the present invention improves thepresentation of the antigen to the immune system, it may be particularlyapplicable to diseases and infections which are generally believed to berecalcitrant to a vaccine approach, e.g. HIV.

In a particularly preferred embodiment, the present invention provides acancer vaccine. Thus the present invention is useful in the treatment oftumors, such as melanoma, prostate, lung, breast and colon cancer andlymphomas.

One of the drawbacks of conventional cancer treatment such aschemotherapy and radiation is that, in destroying the cancer cells, theyalso damage some healthy tissue. Cancer vaccines seek to amelioratethese problems. The present invention is particularly applicable to usewith tumor-associated antigens. Even though the immune system does notrecognise cancer cells as foreign, it has been found that cancer cellsdo carry particular antigens on their surfaces. These may be unique toindividual tumors, shared by several types, or expressed by the normaltissue from which a tumor anises. The present invention is alsoapplicable to antigens associated with the products of genes involved intumor formation, such as oncogenes and tumor suppressor genes. Thepresent invention is further applicable to viruses which have beenassociated with cancer, such a hepatitis B virus, which has long beenimplicated in the development of liver cancer and EBV.

The present invention may be employed following diagnosis of disease orfollowing initial therapy such as surgery or chemotherapy.

Non-limiting examples of tumor antigens which may be employed in thepresent invention will now be discussed below:

Since the cloning of MAGE-1, the first gene reported to encode a humantumor antigen recognized by T cells, molecular identification andcharacterization of tumor antigens has mainly been achieved formelanoma. A major reason for this lies in the difficulty of establishingcell lines in vitro from other types of cancer, such lines beingnecessary to generate tumor-specific CTL lines or clones to be used inthe genetic or biochemical approach aimed at molecularly identifying newcancer antigens. More recently, however, new approaches have allowed thediscovery of new antigens recognized by T cells even in tumors differentfrom melanoma. Examples from the various antigen categories are givenbelow. However, as discussed above, analogs or artificially modifiedepitopes may also be used in the present invention. Other antigens,identified by antibodies, may also be used and a large collection ofthem, as detected by the Serex technology, can be found in the data baseof the Institute for Cancer Research (www.licr.org/SEREX.htm). It is ofnote that many tumor antigens (e.g. MAGE, NY-ESO-1a) are now known to berecognized by both T cells and antibodies in the same cancer patients.In addition, using recent technologies (e.g. subtractive hybridization,representational-difference analysis, microarrays) hundreds of genes arebeing detected which are preferentially expressed or overexpressed inneoplastic cells as compared with normal counterparts or are expressedin metastatic but not in primary, early lesions (e.g. melanoma, breastcancer, lymphoma, etc.). By using appropriate computers algorithms, anumber of new epitopes will be identified that can bind MHC molecules.By applying such approaches, a large array of gene products can bescreened for their potential antigenic function. Immunogenic epitopescan be selected through appropriate functional assays. All these mayalso be useful in the present invention.

Classification of Tumor Antigens Group 1. Class I HLA-RestrictedCancer/Testis Antigens.

A milestone in tumor immunology was certainly the cloning of MAGE-1 andthe subsequent characterization of the first T-cell-defined antigenicepitope a year later. Those findings were rapidly followed by theidentification of new members within this group. The MAGE, BAGE and GAGEfamilies of genes were born. The antigens belonging to this group, nowincluding also NY-ESO-1, were called cancer/testis (CT) antigens fortheir expression in histologically different human tumors and, amongnormal tissues, in spermatocytes/spermatogonia of testis and,occasionally, in placenta. These antigens now represent one of the maincomponents for antitumor vaccine development. CT antigens result fromreactivation of genes normally silent in adult tissues, but that aretranscriptionally activated in some tumors. CT genes are probably themost characterized ones. New genes in the group of CT antigens whichhave been cloned will include (CT9, CT10, LAGE, MAGE-BS, -B6, -C2, -C3and -D, HAGE, SAGE).

Group 2. Class I HLA-Restricted Differentiation Antigens.

These antigens are shared between tumors and the normal tissue fromwhich the tumor arose; most are found in melanomas and normalmelanocytes. Many of these melanocyte lineage-related proteins areinvolved in the biosynthesis of melanin. Epitopes recognized by bothCD8+ and CD4+ T cells can be derived from melanosome proteins.

Group 3. Class I HLA-Restricted Widely Expressed Antigens.

Genes encoding widely expressed tumor antigens have been detected inmany normal tissues as well as in histologically different types oftumors with no preferential expression on a certain type of cancer. Itis possible that the many epitopes expressed on normal tissues are belowthe threshold level for T-cell recognition, while their overexpressionin tumor cells can trigger an anticancer response even by breaking apreviously established tolerance. These widely expressed gene productshave revealed a broad spectrum of mechanisms that are involved ingenerating T-cell-defined epitopes through alterations in genetranscription and translation. To highlight some examples, the epitopeof CEA is derived from a non-AUG-defined alternative ORF, while the RU2gene creates its epitope by reverse strand transcription.

Group 4. Class I HLA-Restricted, Tumor-Specific Antigens.

Unique tumor antigens arise from point mutations of normal genes (like-catenin, CDK4), whose molecular changes often accompany neoplastictransformation or progression. These antigens are thus expressed only inthe individual tumor where they were identified since it is unlikelythat the same mutation may occur in two different neoplasms unless itinvolves genes (e.g. RAS) whose alteration is an obligatory step inneoplastic transformation. In mouse models unique antigens have beenshown to be more immunogenic than the other groups of shared antigens;since unique antigens are responsible of the rejection of tumortransplants in mice, they have been defined as tumor-specifictransplantation antigens (TSTA).

Other tumor-specific but shared antigens have been described which aregenerated by alteration in splicing mechanisms and which occur in tumorbut not in normal cells, as in the case of TRP-2/INT2.

Group 5. Class II HLA-Restricted Antigens.

Stimulation of the CD4+ T helper cells by tumor antigens is consideredto be impaired or absent in cancer patients and this may be the reasonof an insufficient immune response to tumors. Therefore theidentification of tumor antigen epitopes recognized by such lymphocytesis a crucial step in the long sought improvement of antitumor immuneresponse that may result into clinical efficacy. The first epitopepresented by a class II HLA and capable of provoking a CD4+ T-cellresponse was identified in 1994 in melanoma tyrosinase. Then a gap of 4years followed during which only one additional epitope wascharacterized, before other genes encoding class II-restricted peptideswere discovered. However, as the technical and methodological approachesfor identifying CD4+ T-cell epitopes of tumor antigens have becomeavailable, an exponential increase in reporting such epitopes has beenseen. In fact, since 1998 as many as 27 new class II HLA-restrictedepitopes from 14 antigens have been molecularly identified using, amongothers, Ii-cDNA fusion libraries, immunized transgenic mice andbiochemical approaches. It is of note that even class II-restrictedantigens include a subgroup of mutated proteins which, therefore,represent truly tumor-specific antigens.

Group 6. Fusion Proteins.

In several malignancies, particularly in some forms of leukemias, themolecular mechanism of carcinogenesis involves translocation ofchromosomes which results in fusion of distant genes. This often causesthe synthesis of fusion proteins which characterize each type of disease(e.g. bcr-abl, pml-RAR in CML and APL, respectively) and generate newepitopes that can be recognized by T cells, either CD8+ or CD4+ in classI or class II HLA restriction, respectively. Although these epitopesappear to be weakly immunogenic in leukemia patients, some of thesepeptides or proteins can nevertheless be used to pulse dendritic cellsfor vaccination.

Non-limiting examples of tumor antigens which are particularly useful inthe present invention include: MAGE, BAGE and GAGE, CTL recognisingepitopes from tyrosinase, MelanA^(Mart1), gp100^(me17) and gp75^(TRP1).

Selection Gene or Marker

The T cell of the present invention preferably comprises at least onefurther foreign gene which is a selection gene, or a marker gene. As iswell known selection genes may be used to select transformed cells fromnon-transformed cells. Many different selectable markers have been usedsuccessfully in vectors, and these may equally well be used in thepresent invention. Suitable, bacterial or animal antigens are used. In apreferred embodiment, for instance bacterial antigens (e.g.β-galactosidase), bacterial genes or other genes conferring resistanceto antibiotics (e.g. neo, amp, Kan and tet) or a suicide gene (e.g.HSV-Tk).

In a preferred embodiment, the T cell comprises at least a suicide geneas the further foreign antigen. The advantage offered by the use of sucha suicide gene is that the activation of this gene can be controlled bysystemically administering a further substance such as cyclosporin or5′-fluorocytosin. An examples of a suicide gene includes the herpessimplex virus thymidine kinase gene (HSV Tk) which can kill infected andbystander cells following treatment with ganciclovir.

Other suicide genes include, for example, the cytosine deaminase genewhich confers lethal sensitivity to 5-fluorocytosine. and the human cellsurface molecule CD20, which allows killing of the transduced cells byadministration of rituximab (Introna et al. Hum Gene Ther 11:611, 2000).The advantage of CD20 is that being a human molecule it is notimmunogenic.

A particular advantage of the use of a suicide gene is safety in thatthe it can be used for killing the T cell if undesired side effectsoccur.

According to a particularly preferred embodiment of the presentinvention the selection or marker gene is itself immunogenic orexpresses a protein which is immunogenic. The advantage of such a systemis that enables the immunological status of the patient, and thereforethe effectiveness of the vaccination treatment to be monitored. Examplesof such suitable further foreign antigens include HSV-Tk and Neo, bothof which generate an immune response in an immunocompetent patient. Forexample, immunisation of the patient against the first antigen can bemonitored by detecting anti-HSV-Tk cytolytic activity as is described inmore detail below.

For safety reasons, the T cells of the present invention may also begrowth-arrested, e.g. through irradiation or treatment with mitomycin.Such irradiation may be carried out at room temperature at a dose of 100Rad/minute. Cells exposed to such irradiation no longer proliferate, buton account of their mRNA which is still present in the cell, are stillcapable of expressing proteins.

Treatment

This includes any therapeutic application that can benefit a human ornon-human animal. The treatment of mammals is particularly preferred.Both human and veterinary treatments are within the scope of the presentinvention.

Treatment may be in respect of an existing condition or it may beprophylactic. It may be of an adult, a juvenile, an infant, a foetus, ora part of any of the aforesaid (e.g. an organ, tissue, cell, or nucleicacid molecule).

The T cells prepared by the method of the invention may be administeredto a patient suffering from a malignancy.

Generally, in an ex vivo approach the patient will be the same patientfrom whom the treated T cells originated. Examples of malignancies thatmay be treated include cancer of the breast, cervix, colon, rectum,endometrium, kidney, lung, ovary, pancreas, prostate gland, skin,stomach, bladder, CNS, oesophagus, head-or-neck, liver, testis, thymusor thyroid. Malignancies of blood cells, bone marrow cells,B-lymphocytes, T-lymphocytes, lymphocytic progenitors or myeloid cellprogenitors may also be treated.

The tumor may be a solid tumor or a non-solid tumor and may be a primarytumor or a disseminated metastatic (secondary) tumor. Non-solid tumorsinclude myeloma; leukaemia (acute or chronic, lymphocytic or myelocytic)such as acute myeloblastic, acute promyelocytic, acute myelomonocytic,acute monocytic, erythroleukaemia; and lymphomas such as Hodgkin's,non-Hodgkin's and Burkitt's. Solid tumors include carcinoma, coloncarcinoma, small cell lung carcinoma, non-small cell lung carcinoma,renal carcinoma, adenocarcinoma, melanoma, basal or squamous cellcarcinoma, mesothelioma, neuroblastoma, glioma, astrocytoma,medulloblastoma, retinoblastoma, sarcoma, osteosarcoma,rhabdomyosarcoma, fibrosarcoma, osteogenic sarcoma, hepatoma, andseminoma.

Typically the composition of the present invention may be administeredwith a tumor-specific antigen such as antigens which are overexpressedon the surface of tumor cells.

The T cells may be used to treat an ongoing immune response (such as anallergic condition or an autoimmune disease) or may be used to generatetolerance in a patient. Thus the cells of the present invention may beused in therapeutic methods for both treating and preventing diseasescharacterised by inappropriate lymphocyte activity in animals andhumans. The T cells may be used to confer tolerance to a single antigenor to multiple antigens.

Typically, T cells are obtained from the patient or donor and primed asdescribed above before being returned to the patient (ex vivo therapy).

Pharmaceutical Compositions

A pharmaceutical composition is a composition that comprises or consistsof a therapeutically effective amount of a pharmaceutically activeagent. It preferably includes a pharmaceutically acceptable carrier,diluent or excipients (including combinations thereof), Acceptablecarriers or diluents for therapeutic use are well known in thepharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).The choice of pharmaceutical carrier, excipient or diluent can beselected with regard to the intended route of administration andstandard pharmaceutical practice. The pharmaceutical compositions maycomprise as—or in addition to—the carrier, excipient or diluent anysuitable binder(s), lubricant(s), suspending agent(s), coating agent(s),solubilising agent(s).

“Therapeutically effective amount” refers to the amount of thetherapeutic agent which is effective to achieve its intended purpose.White individual patient needs may vary, determination of optimal rangesfor effective amounts of each nitric oxide adduct is within the skill ofthe art. Generally the dosage regimen for treating a condition with thecompounds and/or compositions of this invention is selected inaccordance with a variety of factors, including the type, age, weight,sex, diet and medical condition of the patient, the severity of thedysfunction, the route of administration, pharmacological considerationssuch as the activity, efficacy, pharmacokinetic and toxicology profilesof the particular compound used, whether a drug delivery system is used,and whether the compound is administered as part of a drug combinationand can be adjusted by one skilled in the art. Thus, the dosage regimenactually employed may vary widely and therefore may deviate from thepreferred dosage regimen set forth herein.

Examples of pharmaceutically acceptable carriers include, for example,water, salt solutions, alcohol, silicone, waxes, petroleum jelly,vegetable oils, polyethylene glycols, propylene glycol, liposomes,sugars, gelatin, lactose, amylose, magnesium stearate, talc,surfactants, silicic acid, viscous paraffin, perfume oil, fatty acidmonoglycerides and diglycerides, petroethral fatty acid esters,hydroxymethyl-cellulose, polyvinylpyrrolidone, and the like.

The dosage range required depends on the choice of peptide, the route ofadministration, the nature of the formulation, the nature of thesubject's condition, and the judgement of the attending practitioner.Suitable dosages, however, are in the range of 0.1-100 μg/kg of subject.

A vaccine composition is conveniently in injectable form. Conventionaladjuvants may be employed to enhance the immune response. A suitableunit dose for vaccination is 0.5-5 microgram/kg of antigen, and suchdose is preferably administered 1-3 times and with an interval of 1-3weeks. With the indicated dose range, no adverse toxicological effectswill be observed with the compounds of the invention which wouldpreclude their administration to suitable individuals.

Wide variations in the needed dosage, however, are to be expected inview of the variety of compounds available and the differingefficiencies of various routes of administration. For example, oraladministration would be expected to require higher dosages thanadministration by intravenous injection. Variations in these dosagelevels can be adjusted using standard empirical routines foroptimisation, as is well understood in the art.

Preferred features and embodiment of the present invention will now bedescribed further with reference to the following Examples and Figures.

DESCRIPTION OF THE FIGURES

FIG. 1 presents results of cell lysis experiments using effector cellsisolated from the infused patients against stimulator cells (fullsymbol) and against unrelated targets (empty symbols). Effector cellswere generated by the stimulation of peripheral blood mononuclear cells(PBMC) isolated from 7 treated patients with autologous transduced Tcells (circle) and with allogeneic T cells (square). Transduction wasperformed with vectors encoding the HSV-TK protein and the cell surfacemarker ΔLNGFr (deleted human low affinity nerve growth factor receptor).

FIG. 2 presents results of cell lysis experiments using effector cellsisolated from a treated patient against unmodified cells and cellsexpressing either HSV-TK or ΔLNGFr.

FIG. 3 shows the relation between infusions' time, number of engineeredT cells injected and the development of a transgene-specific immuneresponse. Data points represent the cell infusion responsible (fullsymbols) or not responsible (empty symbols) for the induction of theimmune response.

FIG. 4 presents results of cell lysis experiments using effector cellsisolated from two healthy donors against the stimulator cells (fallsymbol) and against unrelated targets (empty symbols). Effector cellswere generated by the stimulation of PBMC isolated from 2 healthy donorswith autologous transduced T cells (circle) and with allogeneic T cells(square).

FIG. 5 shows a schematic representation of the clinical history andfollow-up of patient UPN-15. The presence (full circle) or the absence(empty circle) of circulating transduced T cells was monitored at theindicated time points by PCR with HSV-TK-specific primers. Thedevelopment of an immune response was monitored by ex vivo stimulationof circulating lymphocytes against autologous-transduced and allogeneicT cells. The patient was considered not immunized (empty stars) orimmunized (full stars) against the transgene depending on theanti-HSV-TK cytolytic activity detected.

FIG. 6 shows the results of ELISPOT experiments using fresh lymphocytesisolated from a treated melanoma patient, against autologous cellsexpressing either MAGE-3 or HSV-Tk and against unmodified autologous EBVand melanoma cell lines.

FIG. 7 shows the results of IFNg release experiments using effectorcells isolated in vitro from a treated melanoma patient, against eitherMAGE-3 transduced and untransduced T cells (i.e. PHA-M3 and PHA)respectively, and autologous and unrelated melanoma cell lines. Effectorcells were generated by the stimulation of PBMC isolated from thetreated patient with autologous MAGE-3 transduced T cells.

EXAMPLES Example 1

The infusion of donor lymphocytes in the context of allogeneic bonemarrow transplantation (allo-BMT) for leukaemia is an establishedstrategy to control tumor relapse and viral infections. To circumventthe inherent risk of graft-vs-host disease (GVHD) BM-donor lymphocytescan be transduced by a suicide gene, conferring sensitivity to a drug,that may allow the selective elimination of the transduced cells forboth therapeutic and safety issues.

Peripheral blood mononuclear cells (PBMC) were isolated by Lymphoprep(Nycomed, Oslo, Norvay) density-gradient centrifugation from all theBM-donors and the healthy controls studied.

Activation. Two different activation signals were used to activate Tcells (hereafter referred to as T blasts): phytoemoagglutinine (PHA) 2μg/ml (Boehringer Mannheim); anti-CD3 mAb (OKT3) 30 ng/ml (Orthoclone,Milan, Italy); OKT3 (30 ng/ml). Activated T cells were cultured at 1×10⁶cells/ml, in RPMI 1640 supplemented with 5% autologous serum in thepresence of hu-r-IL-2 100 U/ml (EuroCetus Italia S.r.l., Milan, Italy).Culture medium was changed every 3-4 days.

Transduction. Two methods of lymphocyte transduction were utilized.1-coculture: Lymphocyte activation was performed on a semiconfluentmonolayer of lethally irradiated packaging cells (100 Grays; GIL RAD,Gilardoni, Mandello, Italy), in tissue culture flasks in the presence ofpolybrene (8 μg/ml). The cocultivation was performed for 72 hours.2-spinoculation: After 3-4 days of activation lymphocytes were recoveredand transduced by 2 cycles of 2 hours-centrifugation in the presence ofcell free retroviral supernatants and polybrene (8 μg/ml).

After the transduction procedures, lymphocytes were harvested and seededin fresh medium (10⁶/ml). Retroviral transduced cells were analyzed forΔLNGFr expression on day 5 by FACS with the mouse-anti-human LNGFR mAb20.4 (ATCC, Rockville, Md.).

Selection. Pure populations of transduced cells were obtained by twoselection methods. Lymphocytes transduced with the SFCMM2 vector,encoding the HSV-TK/neo (TN) fusion protein, a bifunctional proteinconferring both the neomycin resistance and the HSV-TK activity, wereselected by the addition of neomycin (0.8 μg/ml) to the culture medium,Fresh neomycin was added at day 3, 7 and 10 of culture.

Lymphocytes transduced with the SFCMM3 vector, encoding the wild typeHSV-TK and the ΔLNGFr were immunoselected by magnetic sorting with themAb 20.4 and rat anti-mouse-IgG1 coated beads (Dynabeads M-450, DynalA.S. N0212 Oslo, Norway).

Pure populations of transduced cells were frozen and then administeredto the patients or utilized ex vivo as stimulator to monitorantigen-specific immune responses.

Samples of PBMCs were taken from the 7 treated patients, and contactedto either irradiated transduced T cells of the respective BM-donor, orirradiated allogeneic T blasts. After 2 days of culture 10 U/ml of IL2were added. The mixtures were observed for lysis of the stimulatorcells, which indicated that cytotoxic T lymphocytes (CTLs) specific fora transgene product expressed by the transduced T cells or for analloantigen were present in the samples. The lysis assay employed was achromium release assay. The results presented in FIG. 1 show thatcytotoxic T cells with specificity for the transduced targets wereobtained from all the patients. To assess the patients'immunocompetence, specific immune response against allogeneic targetswas tested and detected in all the responders. This suggests thatinfusion of T blasts, transduced under the conditions described above,are able to elicit immune responses against the transduced cells.

Example 2

Further studies were carried out to identify the target antigens of theimmune response. Effector cells derived from one of the patient weretested against autologous T blasts expressing single components of thevector. T blasts expressing HSV-TK were lysed while target cellsexpressing the ΔLNGFr were not recognized at all (FIG. 2). Analogousresults were obtained with effector cells isolated from other patients.

Example 3

The results from Example 1 (i.e. generation of HSV-TK-specific immunity)were analyzed in relation to the time of the infusions and the number ofengineered T cells infused. Time of the infusions, measured as monthsfrom bone marrow transplantation (abscissa) and number of engineered Tcells injected (ordinate), are presented. The development of an immuneresponse against the transduced cells was evaluated by ex vivostimulation of PBLs against autologous-transduced and allogeneic T cellsas described in example 1. In the presence of a positive anti-alloimmune response the patient was considered not immunized (empty symbols)or immunized (full symbols) to the transgene depending on the presenceof a cytolytic activity against the transduced cells. Data pointsrepresent the cell infusion responsible (full symbols) or notresponsible (empty symbols) for the induction of the immune response.For patient UPN-15 two different time points are represented. Theseresults suggested that induction of the immune response by the in vitromanipulated cells was independent of the number of infused cells andstrictly related to the presence of a functional immune system at thetime of the infusion.

Example 4

Further studies were carried out to identify the mechanism utilized bythe transduced T blasts to elicit in vivo a transgene-specific immuneresponse. Samples of PBMCs were taken from 2 healthy donors and werestimulated accordingly to example 1 with either irradiated autologoustransduced T blasts, or irradiated allogeneic T blasts. After 2 days ofculture 10 U/ml of IL2 were added. The mixtures were observed for lysisof the stimulator cells, which indicated that CTLs specific for atransgene product expressed by the transduced T blasts or for analloantigen were present in the samples. The results presented in FIG. 4show that cytotoxic T cells with specificity for the transduced targetswere not obtained from healthy controls. Thus suggesting that in ourexperimental setting transduced T blasts are not able to act asprofessional antigen presenting cells, activating by themselves naive Tcell precursors. An in vivo cross-presentation pathway, mediated byresident APCs, could be responsible for the induction of thetransgene-specific immune response in vivo in the infused patients.

Example 5

Further experiments were carried out to define the cellular mechanismresponsible for the induction of the transgene-specific immune response.The ability to generate effective immune responses and the presence ofcirculating transduced T blasts were evaluated during the follow-up ofpatient UPN-15 (FIG. 5). The presence of circulating transduced T blastswas analysed by PCR with HSV-TK-specific primers, on fresh bloodsamples. Immune reconstitution and HSV-TK-specific immunization wereevaluated as described in example 1. Patient UPN-15 was injected earlyafter transplant in the absence of a functional immune system. Theinfused cells were detected by PCR in the circulation for a long time(FIG. 5 full circles), even when his immune system was fullyreconstituted. Immunological reconstitution was documented by thepresence of an active anti-allo immune repertoire in the absence of anHSV-TK specific immune response (FIG. 5 empty star). Therefore, at thetime of the injection the immunization does not occur because thecapacity of the patient to generate an adequate T cell response isprofoundly compromised in term of both antigen-presenting andantigen-responding capabilities. Later on, when the immune system isfully reconstituted, the circulating transduced T blasts are unable toprovide activation signals, they do not act as APC and therefore,cytotoxic effector cells specific for the transduced cells can not beelicited.

A second injection of HSV-TK-engineered T blasts, performed when theUPN-15's immune system was fully reconstituted, resulted in theinduction of a HSV-TK-specific immunity (FIG. 5 full star) leading tothe destruction of the engineered cells (FIG. 5 empty circle).

Therefore, immunization occurs when T cells manipulated as described inexample 1, are delivered in an immunocompetent melieu. In particular, Tcells act in vivo as a vehicle carrying the antigen in the right placeto induce an immune response. Here, the partial cell death of theinjected cells, mediated by growth factor (i.e. IL2) deprivation, leadsto the release of the carried antigens; this is followed by presentationof the released antigenic material by host APC and activation of naiveHSV-TK-specific T cell precursors.

The foregoing experiments described a new methodology whereby Tlymphocytes can be in vitro modified to become the vehicle for targetantigen delivery in vivo.

Example 6

T lymphocytes can be in vitro modified to become the vehicle for targetantigen delivery in vivo. Autologous lymphocytes from a patient affectedby a MAGE-3 positive melanoma, have been transduced by a retroviralvector encoding the tumor antigen MAGE-3 and the HSV-TK/neo (TN) fusionprotein, a bifunctional protein conferring both the neomycin resistanceand the HSV-TK activity.

The activation transduction and selection procedures utilized were thesame reported in Example 1. At the end of the manipulation process, purepopulations of transduced cells were frozen and then administered to thepatients with an escalating dose protocol of vaccination. The transducedcells were also utilized ex vivo as stimulators to monitorantigen-specific immune responses.

Samples of PBMCs were taken from the melanoma patient before treatmentand after the fifth vaccination. The collected cells were immediatelycontacted to autologous EBV melanoma cells, and EBV transduced witheither MAGE-3 or HSV-TK. The test was performed on ELISPOT-plates coatedwith anti-IFNg mAb. After 24 h of culture the cells were removed and theplates were treated to evaluate the presence of IFNg spots. The resultspresented in FIG. 6 show that IFNg-releasing T cells with specificityfor the transgenes (i.e. MAGE-3 and HSV-TK) were obtained after thevaccination. A significant increase in the frequency of tumor-specific Tcells was also detected. This suggests that infusion of T blasts,transduced under the conditions described above, are able to elicitimmune responses against the transduced cells.

Example 7

PBMCs were taken from the melanoma patient after the vaccinations andcontacted to irradiated MAGE-3-transduced T cells. After 2 days ofculture 10 U/ml of IL2 were added. The mixtures were observed for IFNgrelease in the presence of the stimulator cells, which indicated thateffector cells specific for the MAGE-3 transgene product expressed bythe transduced T cells were present in the samples. The resultspresented in FIG. 7 show that effector T cells with specificity forMAGE-3 and the autologous tumors were obtained. This further confirmsthat infusion of T blasts, transduced under the conditions describedabove, are able to elicit immune responses against the transgeneproducts.

Example 8

To investigate the kinetics of generation of transgenes-specific immuneeffectors, in respect of the dose of transduced cells infused, PBMCswere taken from the melanoma patient before treatment and at severaltime-points of the vaccination protocol (i.e. after 3, 5 and 15vaccination). The collected cells were contacted with either irradiatedMAGE-3-transduced or HSV-TK-transduced T cells. After 2 days of culture10 U/ml of IL2 were added. For each time-point several independentmicro-cultures were plated (ranging from 30 to 48). After several roundsof stimulations the micro-cultures were observed for IFNg release in thepresence of the stimulator cells, which indicates that effector cellsspecific for MAGE-3 or HSV-TK were present in the samples. The resultssummarized in the following table show the existence of a correlationbetween injection of transduced T cells and development of an immuneresponse against the transgenes. A continuous increase of the amount ofMAGE-3 reactive T cell precursors, represented by the number of positivemicro-cultures, was observed

In vivo infusion of MAGE-3-expressing T cells is responsible for theinduction of MAGE-3-specific immune responses. N^(o) of micro-cultureswith specific activity Blood Sample anti-MAGE-3 anti-HSV-TKPre-treatment 0 0 Post-3 3/42 ND Post-5 6/48 48/48 Post-15 30/30  ND ND:not done

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are apparent to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

1-18. (canceled)
 19. A method of monitoring the immune response to an antigen comprising the step of administering a T cell comprising a first antigen and a second antigen capable of raising an immune response and monitoring the response of the immune system against the second antigen as a measure of the response of the immune system to said first antigen.
 20. The method according to claim 19 wherein the first antigen is a tumor antigen.
 21. The method according to claim 19 wherein the first antigen is a bacterial or viral antigen.
 22. The method according to claim 19 wherein the second antigen is a strongly immunogenic antigen.
 23. The method according to claim 19 wherein the second antigen is HSV-Tk or CD20.
 24. A method of loading APCs with an antigen in vivo comprising the step of exposing the APCs to a T cell containing the antigen to obtain APCs loaded with said antigen.
 25. The method according to claim 24 wherein the antigen is a tumor antigen.
 26. The method according to claim 24 wherein the antigen is a bacterial or viral antigen.
 27. method according to claim 24 wherein the T cell contains a marker.
 28. The method according to claim 27 wherein the marker is a marker gene.
 29. The method according to claim 28 wherein the marker is a bacterial resistance gene.
 30. The method according to claim 29 wherein the bacterial resistance gene confers neomycin resistance.
 31. The method according to claim 28 wherein the marker is a further antigen.
 32. The method according to claim 27 wherein the marker is HSV-Tk or CD20.
 33. (canceled)
 34. The method according to claim 24 wherein the T cell expresses at least one of the following markers: HLA-I, HLA-11, CD80, CD86, CD27, CD40L, CD62L, CCR7, CD54 and CD25.
 35. A method of obtaining a T cell for use in the method of claim 18 comprising isolating a T cell; activating the T cell; culturing the T cell; and introducing an antigen into the T cell.
 36. The method according to claim 35 wherein the T cell is transduced with the antigen.
 37. The method according to claim 35 wherein the T cell is activated with phytoemoagglutinine, anti-CD3 monoclonal antibody, or anti-CD3/CD28 monoclonal antibody-coated beads.
 38. The method according to claim 35 wherein the T cell is cultured in the presence of growth factors.
 39. The method according to claim 35 wherein the growth factors include hu-r-IL-2.
 40. The method according to claim 35 wherein the T cell is cultured in a culture media which comprises 5% autologous serum.
 41. The method according to claim 35 wherein the T cell is cultured at 1×10⁶ cells/ml.
 42. A T cell obtainable by the process of claim
 28. 43. A T cell loaded with antigen. 44-45. (canceled)
 46. A method for the treatment or presentation of a tumor or infection comprising administering an effective amount of a T cell as defined in claim 42 for the preparation of a medicament for the treatment or prevention of a tumor, or infection to a patient in need of the same. 